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EC number: 280-163-4 | CAS number: 83027-61-6
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 17 August 2017 (Seeding of the cells, 1st experiment of h-CLAT) - 04 April 2018 (study completion date)
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of dendritic cells
- Justification for non-LLNA method:
- The OECD TG 442 E may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
Test material
- Reference substance name:
- Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[ethyltoluenesulphonate]
- EC Number:
- 280-163-4
- EC Name:
- Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[ethyltoluenesulphonate]
- Cas Number:
- 83027-61-6
- Molecular formula:
- C32H28N2Na2O8S2
- IUPAC Name:
- Reaction mass of disodium 2-ethyl-3-({4-[(2-ethyl-6-methyl-3-sulfonatophenyl)amino]-9,10-dioxo-9,10-dihydroanthracen-1-yl}amino)-4-methylbenzene-1-sulfonate, disodium 4-ethyl-3-({4-[(2-ethyl-6-methyl-3-sulfonatophenyl)amino]-9,10-dioxo-9,10-dihydroanthracen-1-yl}amino)-2-methylbenzene-1-sulfonate and disodium 4-ethyl-3-({4-[(6-ethyl-2-methyl-3-sulfonatophenyl)amino]-9,10-dioxo-9,10-dihydroanthracen-1-yl}amino)-2-methylbenzene-1-sulfonate
- Test material form:
- solid: particulate/powder
Constituent 1
In vitro test system
- Details on the study design:
- Cell line: THP-1 cells
The human monocytic leukemia cell line was obtained from “American Type Culture Collection, Manassas, USA” (ATCC, TIB202).
Results and discussion
- Positive control results:
- The positive control used was 1- chloro-2,4-dinitrobenzene (DNCB, CAS no.: 97-00-7), 4.0 µg/mL in 0.2% DMSO in culture medium. The positive and negative and vehicle control data is comparable to the historical data. The results can be seen in Table 2.
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- other: Experiment 4
- Parameter:
- other: Relative fluorescence intensity (RFI) %
- Remarks:
- RFI CD86
- Value:
- 150
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Negative activation of dendritic cells at concentrations of 78-279 µg/mL
- Remarks:
- viability ≥50%
- Key result
- Run / experiment:
- other: Experiment 4
- Parameter:
- other: Relative fluorescence intensity (RFI) %
- Remarks:
- RFI CD54
- Value:
- 200
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Positive activation of dendritic cells at concentrations at concentrations of 233 and 279 µg/mL
- Remarks:
- viability ≥50%
- Key result
- Run / experiment:
- other: Experiment 5
- Parameter:
- other: Relative fluorescence intensity (RFI) %
- Remarks:
- RDI CD86
- Value:
- 150
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Negative activation of dendritic cells at concentrations of 78-279 µg/mL
- Remarks:
- viability ≥50%
- Key result
- Run / experiment:
- other: Experiment 5
- Parameter:
- other: Relative fluorescence intensity (RFI) %
- Remarks:
- RFI CD54
- Value:
- 200
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Positive activation of dendritic cells at concentrations of 194, 233, 279 µg/mL
- Remarks:
- viability ≥50%
- Other effects / acceptance of results:
- DEMONSTRATION OF TECHNICAL PROFICIENCY:
Acceptance criteria
- A tested concentration is not to be further evaluated when relative viability is less than 50%.
- Cell viability of vehicle control cells must yield at least 90%.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should be over the positive criteria (CD86≥ 150% and CD54 ≥ 200%) and cell viability should be ≥ 50%.
- In the negative control (LA), RFI values of both CD86 and CD54 should not exceed the positive criteria (RFI CD86< 150% and RFI CD54 < 200%) and cell viability should be ≥ 50%.
- For all vehicle controls, the MFI ratio of both CD86 and CD54 to isotype controls should be ≥ 105%.
- The reactivity check of new thawed cells should produce the following result: - Positive response in CD86 and CD54 for NiSO4 and DNCB - Negative response in CD86 and CD54 for LA.
- In addition, positive, negative and vehicle control data should lie within the range of the historic data
Evaluation of results
- A test substance is predicted to activate monocytic THP-1 cells when CD86 expression is increased ≥ 150% and/or CD54 expression increased ≥ 200% at any concentration in relation to vehicle control that do not reduce viability below 50% and reproduced in the same cell surface marker in at least two independent experiments.
- A test substance is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (= 5000 µg/mL for the vehicle culture medium or 1000 µg/mL for 0.2% DMSO in culture medium) or up to the cytotoxicity limit (viability less than 90% at the highest concentration tested).
- To be relevant for evaluation, the cell viability must be more than 50% in at least four tested concentrations of an experiment.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: Please refer to the historical control data in the 'Attached background material' section
Any other information on results incl. tables
Preliminary cytotoxicity assessment
The CV75 value (=estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration-response curve to be 162 µg/mL.
Experiment 1: The concentrations selected (204 -739 µg/mL) were too high based on the results of a not assessable 1st pre-test (cytotoxicity below 50% was observed in all concentrations) and is not useful for evaluation and not included in the report. A 2nd pre-test was performed.
Experiment 2 : Concentration range selected was 54 -194 µg/mL. The experimen twas considered invalid due to a technical error and is not included in the report.
Experiment 3: This was carried out under the same conditions as the 2nd experiment. There was no cytotoxicity below 50% and no positive response (RFI CD86 ≥ 150% and/or CD54 ≥200%) was observed in the 3rd experiment, further experiments were carried out using the concentration range shown in Table 3. Calculation of the EC15 0% (the concentration resulting in a RFI of 150%) for CD86 and the EC200% (the concentration resulting in a RFI of 200%) for CD54 was not applicable.
Experiment 4: The calculation of the EC150% (the concentration resulting in a RFI of 150%) for CD86 was not applicable. The EC200% (the concentration resulting in a RFI of 200%) for CD54 was calculated by linear regression from the results of the 194 µg/mL and the 233 µg/mL concentration to be 203 µg/mL.
Experiment 5: The calculation of the EC150% (the concentration rsulting in a RFI of 150%) for CD86 was not applicable. The EC200% (the concentration resulting in a RFI of 200%) for CD54 was calculated by linear regression from the results of the 162 µg/mL and the 194 µg/mL concentration to be 188 µg/mL.
Table 2: Summary of h-CLAT Main Experiments: mean values of RFI CD86, RFI CD54 and relative viability. 2 valid and evaluable experiments (4thand 5th) were performed. The 1stand3rdexperiments are valid but not useful for evaluation and the 2ndexperiment was invalid and is not included in the report. |
|||||||
4thexperiment |
5thexperiment |
||||||
Concentration (test substance)
[µg/mL) |
RFI CD86
Mean [%] |
RFI CD54
Mean [%] |
Viability
Relative viability [%] |
Concentration (test substance)
[µg/mL) |
RFI CD86
Mean [%] |
RFI CD54
Mean [%] |
Viability
Relative viability [%] |
78 |
58 |
85 |
100 |
78 |
66 |
139 |
100 |
94 |
57 |
76 |
100 |
94 |
58 |
117 |
100 |
112 |
60 |
112 |
100 |
112 |
61 |
144 |
100 |
135 |
62 |
103 |
100 |
135 |
62 |
131 |
100 |
162 |
62 |
125 |
100 |
162 |
65 |
174 |
99 |
194 |
88 |
166 |
97 |
194 |
92 |
205 |
96 |
233 |
127 |
311 |
90 |
233 |
118 |
370 |
90 |
279 |
137 |
451 |
77 |
279 |
138 |
541 |
80 |
VC |
100 |
100 |
100 |
VC |
100 |
100 |
100 |
LA 1000 µg/mL |
77 |
102 |
100 |
LA 1000 µg/mL |
70 |
132 |
100 |
DNCB 4 µG/mL |
284 |
1222 |
70 |
DNCB 4 µG/mL |
285 |
555 |
84 |
RFI above 150% (CD86) or 200% (CD54) with relative viability ≥50% are indicated in bold. VC: vehicle (culture medium); LA : lactic acid, negative control; DNCB : 1 -chloro-2, 4 -dinitrobenzene, positive control |
- The 3rd main experiment met the “borderline”-criteria at one concentration. The 4th and 5th experiments were unambigious positive in CD54.
- If the borderline criteria, defined as stated above, would be applied, these results would be considered ambiguous. A differently defined borderline range may yield different outcomes – no borderline range has yet been defined and assigned to the respective OECD test guideline.
Applicant's summary and conclusion
- Interpretation of results:
- other: positive prediction of skin sensitisation in h-CLAT assay
- Conclusions:
- Based on the observed results and applying the evaluation criteria in accordance with OECD Guideline 422 E, after 24-hours of exposure, Acid Blue 204 induced CD54 expression in THP-1 cells affording at least 50% viability in at least two independent experiments. Therefore, it can be concluded that Acid Blue 204 induces dendritic cell activation and is therefore predicted to be a skin sensitiser.
- Executive summary:
The skin sensitising potential of the test substance, Acid Blue 204, was determined via the in vitro Human Cell Line Activation Test (h-CLAT) that evaluates that ability of Acid Blue 204 to induce the expression of cell membrane markers (CD86 and CD54) and thus activate dendritic cells. The study was conducted following the OECD Guideline for Testing of Chemicals TG. 442E, adopted July 2016 (‘In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)’)
Main Assay
The test substance, Acid Blue 204 was weighed and topped up with culture medium to achieve the required 2x concentration of the high concentration, Further concentrations were prepared as 2x concentrations by serial 1:1.2 dilution. The test substance was incubated with the human monocytic leukemia cell line THP-1 for ca. 24 hours at 37°C and membrane marker expression (CD86 / CD54) was measured by flow cytometry.
In order to determine the concentrations suitable for the main experiment, two pre-tests (non-GLP) were performed. Cells were exposed to several concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 for the 2ndpre-test was 162 µg/mL3, determined by linear regression from the concentration response curve.
In the main test after 24-hour exposure THP-1 cells were stained with FITC labeled anti-humanCD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry. A total of 2 valid and evaluable experiments were performed (4th and 5th experiment). At concentrations used in the main experiment the test substance was soluble in culture medium (2 x stock preparations and final concentrations) and no precipitates were observed at any concentration after 24 hours.
The concentration range (204 -730 µg/mL) used in Experiment 1 was considered too high and was considered not useful for evaluation, therefore a 2nd pre-test was performed. The second experiment (54 -194 µg/mL) was considered invalid due to a technical error. Experiment 1 and 2 are not included in the report. Experiment 3 was conducted under the same conditions as Experiment 2. The calculation of the EC150% for CD86 and the EC200% for CD54 was not applicable. Experiment 4 and 5 were conducted using a concentration range of 78 -279 µg/mL. In Experiment 4, the calculation of the EC150% for CD86 was not applicable and the EC200% for CD54 was calculated by linear regression from the results of the 194 µg/mL and the 233 µg/mL concentration to be 203 µg/mL. In Experiment 5, the calculation of the EC150% for CD86 was not applicable. The EC200% for CD54 was determined by linear regression from the results of the 162 µg/mL and the 194 µg/mL concentration to be 188 µg/mL. The acceptance criteria were met in all experiments and the results for the positive, negative and vehicle controls are comparable with historic data.
Conclusion
Based on the observed results and applying the evaluation criteria in accordance with OECD Guideline 422 E, after 24-hours of exposure, Acid Blue 204 induced CD54 expression in THP-1 cells affording at least 50% viability in at least two independent experiments. Therefore, it can be concluded that Acid Blue 204 induces dendritic cell activation and is therefore predicted to be a skin sensitiser.
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